Although identified relatively recently, miRNAs have been recognized as one of the major regulatory gene families that utilize identical cellular enzymes/pathways as siRNA with similar mechanisms leading to translational repression of target mRNA. Emerging evidences strongly suggest a crucial role played by miRNAs in various cellular mechanisms including cancer pathogenesis. Micro-RNA genes are transcribed, generally by RNA polymerase II (Pol II), generating the primary miRNA (pri-miRNA). In the nucleus, the RNase III endonuclease Drosha cleaves the pri-miRNA to produce ˜70-nucleotide precursor miRNA (pre-miRNA). Exportin-5 transports the pre-miRNA into the cytoplasm, where it is cleaved by another RNase III endonuclease, Dicer, to a ˜21-nucleotide miRNA duplex. These miRNA molecules are loaded into RNA-induced silencing complex (RISC) which will help knockdown target messenger RNA.
In the recent past, a particularly important role for miRNAs in cancer pathogenesis has emerged. Virtually all examined tumors globally displayed abnormal miRNA expression contributing to cellular transformation and tumorigenesis. Due to their importance in controlling various cellular functions related to cell division and differences and their dramatic alterations in cancer, potential therapeutic approaches have been envisaged. Several lines of evidences suggest that miRNA replacement represents a viable and efficacious strategy. Although specific miRNAs are often over-expressed in cancer cells, most miRNAs are down regulated in tumors.
Silencing of an abnormally elevated miRNA or enforced expression of under expressed miRNA in cancer are ideal targets for gene correction therapy. Small interfering RNAs and miRNAs share similar endogenous biological processing pathways. miRNA expression and processing can be regulated through similar mechanism that controls siRNA. The similarities between miRNA and siRNA suggest that miRNAs also have the potential to affect epigenetic mechanisms including methylation and histone deacetylation leading to diseases like cancers caused by somatic gene aberrations.
Global miRNA profile studies revealed several specific miRNAs with altered expressions contributing to hepatocyte transformation and metastasis. The role of miRNAs in human cancer is further supported by the fact that >50% of miRNA genes are located at fragile genomic loci prone to deletion or amplification that are frequently altered in human cancers. Correcting the altered micro-RNA genes in liver cancer may constitute an important therapeutic approach. Significant under expression of miRNA-101, is a molecular lesion associated with tumor progression. Genomic loss of miRNA-101 in cancer leads to over expression of EzH2 and concomitant dysregulation of epigenetic pathways, resulting in cancer progression.
miRNAs' have a broad specificity as they do not require a perfect match with the complementary sequence of their target mRNA. This creates a possibility of unintended, non-specific targeting of genes. But the fact that the miR-101 silences the expression of several tumor promoting genes such as COX-2, PKCa, suggests that its enforced expression in cancer cells will knockdown tumor promoting genes. Even though the role of miR1O1 has been studied in several solid epithelial malignancies, relatively little is known about its involvement in the progression of liver cancer.
Enhancer of zeste homolog 2 (EzH2) is a mammalian histone methyltransferase that contributes to the epigenetic silencing of target genes and regulates the survival and metastasis of cancer cells. Of the 34 miRNAs predicted to regulate EzH2, only miR-101 is found to have a strong negative association with cancer progression from benign to localized disease to metastasis. Analysis of human prostate tumors revealed that miR-101 expression decreases during cancer progression, paralleling an increase in EzH2 expression. Expression and function of EzH2 in cancer cell lines are inhibited by microRNA-101 (miR-101).
EzH2 is over expressed in aggressive solid tumors by mechanisms that remain unclear. EzH2 is a catalytic sub-unit of polycomb group of repressor proteins which catalyze methylation of chromatin leading to transcriptional silencing of several tumor suppressor genes or anti-oncogenes. The loss of miR-101 and concomitant elevation of EzH2 is most pronounced in metastatic cancer, suggesting that the loss of miR-101 may represent a progressive molecular lesion in the development of more aggressive disease. Approaches to reintroduce miR-101 into tumors may have therapeutic benefit by reverting the epigenetic program of tumor cells to a more normal state.
Hepato-cellular cancer (HCC) is commonest primary liver cancer accounting for roughly 90% of this class of malignancy with poor prognosis due to rapid spread. Inadequacy of current therapies and presentation of patients with advanced diseases have meant that the treatment is generally palliative and prognosis is extremely grave. Early detection combined with novel effective combinatorial therapies are needed for improving management of HCC patients. The increasing evidences have indicated that miR-101 was regarded as a metastatic determinant and a key component in tumor metastasis in several malignancies including liver cancer.
One of the most difficult challenges impeding the advancement of RNA/-based HCC therapy is efficient and safe delivery of effecter sequences. Ideally, vectors deliver silencing molecules selectively to most if not all the malignant hepatocytes. Viral vectors are generally more efficient vehicles in vivo than non viral vectors. Viral vectors have been successfully used for enforced expression of miRNAs in various gene therapy studies established their delivery and efficacy.
Although viral vectors permit the efficient delivery and stable expression of miRNA, establishment of safety, efficacy and potent gene silencing are crucial ingredients for selecting the viral delivery vehicle. A key area of research in the use of RNA/for clinical applications is the development of a safe delivery method, which to date has involved mainly viral vector systems similar to those suggested for gene therapy.
Adeno-associated virus (AAV) is one of most promising vectors for gene therapy. The recombinant AAV (rAAV) provides a nonpathogenic and latent infection by integrating into the host genome; it also shows high transduction efficiency of both dividing and non-dividing cells and tissues with persistent transgene expression. Such recombinant AAV's have the advantage of exhibiting modified tropism, (i.e., being highly selective with respect to the tissues it infects), as well as having a higher rate of transduction efficiency when compared to native AAV. Adeno-associated virus (AAV) is currently being tested in several human gene therapy trials because of its several unique features that distinguish it from other gene therapy vectors. These features include (i) a broad host range; (ii) lack of cell-mediated immune response against the vector; (iii) ability to integrate into a host chromosome or persist episomally, thereby creating potential for long-term expression; (iv) minimal influence on changing the pattern of cellular gene expression and the like.
Hence there is a need for a treatment for liver cancer incorporating the expression of pre-miR-101.